Bande et al. BMC Res Notes (2015) 8:452 DOI 10.1186/s13104-015-1420-5
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Detection of circulating melanoma cells in choroidal melanocytic lesions Manuel F. Bande1*, Maria Santiago1, Laura Muinelo‑Romay2, Maria Jose Blanco1, Purificacion Mera1, Carmela Capeans1, Maria Pardo3 and Antonio Piñeiro1
Abstract Purpose: To detect and quantify circulating tumour cells (CTCs) in peripheral blood of patients with uveal melanoma primary non-metastatic tumours, and to analyze the possible relationship between CTCs and clinical risk factors. Methods: Prospective study with two clinical groups: 4 patients diagnosed with choroidal nevus and 8 patients with choroidal melanoma prior to treatment. A single sample of 7.5 mL of peripheral blood was taken and the CTCs were isolated using a CellSearch system that captures positive cells for the CD146 antigen (MUC18). Results: None of the patients with choroidal nevus showed CTCs in peripheral blood. More than one CTC/7.5 mL was detected in 50 % of patients with choroidal melanoma prior to treatment. The higher level of CTC cells in periph‑ eral blood (3/7.5 mL) was detected in the patient with the larger choroidal melanoma which also presented extrascle‑ ral extension and epithelioid pathology. Conclusion: Performing an analysis with the CellSearch system allows to quantify the choroidal melanoma CTCs in peripheral blood. This finding highlights the potential usefulness of this technique to achieve the correct stratification and monitoring of the treatment. Background Despite the successful treatment of uveal melanoma (UM) primary tumors, patients remain at risk of developing metastases for more than 20 years after the initial diagnosis. In the Collaborative Ocular Melanoma Study (COMS), Kaplan–Meier analyses estimated that the 2-, 5-, and 10-year metastasis rates were 10, 25, and 34 %, respectively. However, only 0.24 % of patients exhibited detectable metastases at the time of diagnosis [1]. The CellSearch system (Veridex) was developed to identify and quantify CTCs in the peripheral blood by immunomagnetic isolation and inmunohistochemical detection. This platform obtained the Food and Drug Administration (FDA) clearance for the CTC enumeration in patients with breast, colon, or prostate cancers [2, 3]. Despite that CellSearch system has been previously *Correspondence:
[email protected] 1 Ocular Oncology Unit, Servizo de Oftalmoloxía, Complexo Hospitalario Universitario de Santiago, Universidade de Santiago de Compostela, Santiago de Compostela, Spain Full list of author information is available at the end of the article
used in the detection of CTC in patients with metastatic uveal melanoma [4]; this technology has not been tested for non-metastatic/localized UM and choroidal nevi until now. Our group investigated the possibility of detecting and quantifying CTCs using the semiautomatic CellSearch system in the peripheral blood of patients CTCs in the patients with primary/localized UM. Further, in this preliminary study we examined the relationship between the presence of CTCs, clinical parameters and disease-free survival.
Methods 12 Patients (8 UM and 4 choridal nevus) diagnosed at the Ocular Oncology Unit (Sevicio de Oftalmología, Santiago de Compostela, Spain) were included in the study after informed consent according to the Declaration of Helsinky. This study was also approved by the Comité Ético de Investigación Clínica de Galicia. Peripheral blood (7.5 mL, CellSave preservative tube, Veridex) was extracted at identical venipuncture points from each patient. Melanoma
© 2015 Bande et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Bande et al. BMC Res Notes (2015) 8:452
cells were detected by the CellSearch system (Veridex, USA) as previously described [5, 6]. Briefly, melanoma cells were isolated with magnetic beads coated with antiCD146 antibody. Then the CD146-expressing cells were stained with the fluorescent nucleic acid dye 4′,6-diamidino-2-phenylindole dihydrocloride (DAPI) and with a combination of fluorescent antibodies against high-molecular-weight melanoma-associated antigen (MEL), CD34 and CD45 to distinguish melanoma cells from leukocytes and endothelial cells (Fig. 1). Cell were considered CTCs when they have oval morphology and were positive for DAPI, MEL and negative for CD34 and CD45. Absence of metastatic melanoma at the time of blood sampling was confirmed by clinical evaluation, routine biochemistry and liver ultrasonography in all patients. The comparisons were done by using the Mann–Whitney U test. The correlations were done by Pearson’s correlation analysis.
Results Among the eight patients with non-treated choroidal melanoma, 50 % displayed more than one CTC per
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7.5 mL of blood. No significant correlation was shown between the CTC positivity and clinicopathological parameters, including the diameter of the largest tumor basal (LBD), the height of the tumor. The average patient follow-up was 25 months (min 16; max 27 months). The most important descriptive observation that emerged from this preliminary study was obtained from the largest choroidal melanoma, which demonstrated extrascleral extension (patient 1, Table 1). This patient presented the greatest number of CTCs and he was the only patient who presented metastatic liver disease at 12 months follow up. Moreover, four choroidal nevi (
